The present invention relates to three novel peptidoglycan recognition binding proteins expressed by keratinocytes, wound-healing tissues and chondrosarcoma tissue. More specifically, isolated nucleic acid molecules are provided encoding human peptidoglycan recognition protein-related proteins, referred to herein as PGRP-K (Keratinocytes), PGRP-W (Wound-healing), and PGRP-C (Chondrosarcoma) of FIGS. 1A-B, FIGS. 2A-C, and FIG. 3, respectively, each having homology to both human peptidoglycan recognition protein (PGRP) as well as murine Tag-7. PGRP-K, PGRP-W, and PGRP-C polypeptides are also provided. Further provided are vectors, host cells and recombinant methods for producing the same. The invention also relates to both the inhibition and enhancement of activities of PGRP-K, PGRP-W, and PGRP-C polypeptides and diagnostic methods for detecting PGRP-K, PGRP-W, and PGRP-C gene expression.
Peptidoglycan, as well as Lipopolysaccharide (LPS), is a surface component of many bacteria which illicit a wide range of physiological and immune responses in humans. Specifically, peptidoglycan has been shown to manifest itself clinically by reproducing most of the symptoms of bacterial infection, including fever, acute-phase response, inflammation, septic shock, leukocytosis, sleepiness, malaise, abcess formation, and arthritis (see Dziarski et al., JBC, 273 (15): 8680 (1998)). Furthermore, the type of peptidoglycan (i.e.xe2x80x94the specific stereoisomers or analogs of muramyl dipeptide, N-acetylglucosaminyl-beta(1-4)-N-acteylmuramyl tetrapeptides, etc.), were shown to elicit a broad range of activities, including exhibiting greater pyrogenicity, inducing acute joint inflammation, stimulating macrophages, and causing hemorrhagic necrosis at a primed site (See Kotani et al., Fed Proc, 45(11): 2534 (1986)).
It has been demonstrated in humans that a lipopolysaccharide binding protein exists that was discovered as a trace plasma protein (See Schumann et al., Science, 249(4975):1429 (1990)). It is thought that one of the modes of action by which this lipopolysaccharide binding protein functions is by forming high-affinity complexes with lipopolysaccharide, that then bind to macrophages and monocytes, inducing the secretion of tumor necrosis factor. Dziarski and Gupta (See Dziarski et al., JBC, 269(3): 2100 (1994)) demonstrated that a 70 kDa receptor protein present on the surface of mouse lymphocytes served to bind heparin, heparinoids, bacterial lipoteichoic acids, peptidoglycan, and lipopolysaccharides.
Recently, Dziarski et al. demonstrated that the CD14, a glycosylphosphatidylinositol-linked protein present on the surface of macrophage and polymorphonuclear leukocytes, bound peptidoglycan and lipopolysaccharide. Furthermore, the binding affinity of CD14 for lipopolysaccharide was significantly increased in the presence of a LPS-binding protein present in plasma. It is thought that the LPS-binding protein functions as a transfer molecule, whereby it binds LPS and presents it to the CD14 receptor (See Dziarski et al., JBC, 273(15): 8680 (1998)).
Yoshida et al. isolated a peptidoglycan binding protein from the hemolymph of the Silkworm, Bombyx mori, using column chromatography. This protein was found to have a very specific affinity for peptidoglycan (See Yoshida et al., JBC, 271(23): 13854 (1996)). Additionally, Kang et al. recently cloned a peptidoglycan binding protein from the moth Trichoplusia ni. The peptidoglycan binding protein was shown to bind strongly to insoluble peptidoglycan (See Kang et al., PNAS, 95(17): 10078 (1998)). In this study the peptidoglycan binding protein was upregulated by a bacterial infection in T. ni. The insect immune system is regarded as a model for innate immunity. Thus, Kang et al were able to clone both mouse and human homologs of the T. ni peptidoglycan binding protein. All of these peptidoglycan binding proteins shared regions of homolgy, as well as four conserved cysteine residues which may function in the tertiary structure of the protein, possibly in helping to form binding domains. Given that peptidoglycan is an integral component of bacterial cell walls, and that it induces many physiological responses from cytokine secretion to inflammation and macrophage activation, it appears as if this family of proteins may be a ubiquitous group involved in the binding and recognition of peptidoglycan, the presentation of antigens (e.g., cell wall components, etc.), and the activation of the immune system, such as the secretion of cytokines, such as TNF.
TNF is noted for its pro-inflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells (Pober, J. S. et al., J. Immunol. 136:1680 (1986)), increased adherence of neutrophils and lymphocytes (Pober, J. S. et al., J. Immunol. 138:3319 (1987)), and stimulation of the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J. Exp. Med. 166:1390 (1987)).
Recent evidence implicates TNF in the pathogenesis of many infections (Cerami, A. et al., Immunol. Today 9:28 (1988)), immune disorders, neoplastic pathology, e.g., in cachexia accompanying some malignancies (Oliff, A. et al., Cell 50:555 (1987)), and in autoimmune pathologies and graft-versus host pathology (Piguet, P.-F. et al., J. Exp. Med. 166:1280 (1987)). The association of TNF with cancer and infectious pathologies is often related to the host""s catabolic state. A major problem in cancer patients is weight loss, usually associated with anorexia. The extensive wasting which results is known as xe2x80x9ccachexiaxe2x80x9d (Kern, K. A. et al. J. Parent. Enter. Nutr. 12:286-298 (1988)). Cachexia includes progressive weight loss, anorexia, and persistent erosion of body mass in response to a malignant growth. The cachectic state is thus associated with significant morbidity and is responsible for the majority of cancer mortality. A number of studies have suggested that TNF is an important mediator of the cachexia in cancer, infectious pathology, and in other catabolic states.
TNF is thought to play a central role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock (Michie, H. R. et al., Br. J Surg. 76:670-671 (1989); Debets, J. M. H. et al., Second Vienna Shock Forum, p.463-466 (1989); Simpson, S. Q. et al., Crit. Care Clin. 5:27-47 (1989)), including fever, malaise, anorexia, and cachexia. Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF (Kombluth, S. K. et al, J. Immunol. 137:2585-2591 (1986)) and other cytokines. Because TNF could mimic many biological effects of endotoxin, it was concluded to be a central mediator responsible for the clinical manifestations of endotoxin-related illness. TNF and other monocyte-derived cytokines mediate the metabolic and neurohormonal responses to endotoxin (Michie, H. R. et al., N. Eng. J. Med. 318:1481-1486 (1988)). Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release (Revhaug, A. et al., Arch. Surg. 123:162-170 (1988)). Elevated levels of circulating TNF have also been found in patients suffering from Gram-negative sepsis (Waage, A. et al., Lancet 1:355-357 (1987); Hammerle, A. F. et al., Second Vienna Shock Forum p. 715-718 (1989); Debets, J. M. H. et al., Crit. Care Med. 17:489-497 (1989); Calandra, T. et al., J. Infec. Dis. 161:982-987 (1990)).
Passive immunotherapy directed at neutralizing TNF may have a beneficial effect in Gram-negative sepsis and endotoxemia, based on the increased TNF production and elevated TNF levels in these pathology states, as discussed above. Antibodies to a xe2x80x9cmodulatorxe2x80x9d material which was characterized as cachectin (later found to be identical to TNF) were disclosed by Cerami et al. (EPO Patent Publication 0,212,489, Mar. 4, 1987). Such antibodies were said to be useful in diagnostic immunoassays and in therapy of shock in bacterial infections. Rubin et al. (EPO Patent Publication 0,218,868, Apr. 22, 1987) disclosed monoclonal antibodies to human TNF, the hybridomas secreting such antibodies, methods of producing such antibodies, and the use of such antibodies in immunoassay of TNF. Yone et al. (EPO Patent Publication 0,288,088, Oct. 26, 1988) disclosed anti-TNF antibodies, including mAbs, and their utility in immunoassay diagnosis of pathologies, in particular Kawasaki""s pathology and bacterial infection. The body fluids of patients with Kawasaki""s pathology (infantile acute febrile mucocutaneous lymph node syndrome; Kawasaki, T., Allergy 16:178 (1967); Kawasaki, T., Shonica (Pediatrics) 26:935 (1985)) were said to contain elevated TNF levels which were related to progress of the pathology (Yone et al., supra).
Accordingly, there is a need to provide molecules that are involved in pathological conditions. Such novel proteins could be useful in augmenting the immune system in such areas as immune recognition, antigen presentation, and immune system activation. Antibodies or antagonists directed against these proteins may be useful in reducing or eliminating disorders associated with TNF and TNF-like cytokines, such as endotoxic shock and auto-immune disorders, for example.
The present invention provides isolated nucleic acid molecules comprising polynucleotides encoding three novel proteins that are structurally similar to a human Peptidoglycan Recognition Protein and murine Tag-7, and are believed to have similar biological effects and activities. The cytokines are named PGRP-K, PGRP-W. and PGRP-C, and the invention includes PGRP-K, PGRP-W, and PGRP-C polypeptides having at least a portion of the amino acid sequence in FIGS. 1A-B (SEQ ID NO:2), in FIGS. 2A-C (SEQ ID NO:4), and/or FIG. 3 (SEQ ID NO:6) or amino acid sequence encoded by the cDNA clones deposited on Dec. 23, 1998, assigned ATCC number 203564; Dec. 23, 1998, assigned ATCC number 203563; and Mar. 20, 1998, assigned ATCC number 209683, respectively. The nucleotide sequence determined by sequencing the deposited PGRP-K clone, which is shown in FIGS. 1A-B (SEQ ID NO:1), contains an open reading frame encoding a complete polypeptide of 243 amino acid residues including an N-terminal methionine, a predicted PGRP-like domain of about 83 amino acid residues, and a deduced molecular weight for the complete protein of about 27 kDa.
The nucleotide sequence determined by sequencing the deposited PGRP-W clone, which is shown in FIGS. 2A-C (SEQ ID NO:3), contains an open reading frame encoding a complete polypeptide of 368 amino acid residues including an N-terminal methionine, a predicted PGRP-like domain of about 83 amino acid residues, and a deduced molecular weight for the complete protein of about 40 kDa.
The nucleotide sequence determined by sequencing the deposited PGRP-C clone, which is shown in FIG. 3 (SEQ ID NO:5), contains an open reading frame encoding a complete polypeptide of 196 amino acid residues including an N-terminal methionine, a predicted PGRP-like domain of about 83 amino acid residues, and a deduced molecular weight for the complete protein of about 21 kDa.
Thus, one aspect of the invention provides isolated nucleic acid molecules comprising polynucleotides having nucleotide sequences selected from the group consisting of: (a) a nucleotide sequence encoding a full-length PGRP-K, PGRP-W, or PGRP-C polypeptide having the complete amino acid sequence in FIGS. 1A-B (SEQ ID NO:2), in FIGS. 2A-C (SEQ ID NO:4), or in FIG. 3 (SEQ ID NO:6), respectively, or as encoded by the cDNA clones contained in the ATCC Deposit number 203564, deposited on Dec. 23, 1998; ATCC Deposit number 203563, deposited on Dec. 23, 1998; and ATCC Deposit number 209683, deposited Mar. 20, 1998, respectively.; (b) a nucleotide sequence encoding the predicted PGRP-like domain of the PGRP-K polypeptide having the amino acid sequence at positions 24 to 107 in FIGS. 1A-B (SEQ ID NO:2), the predicted PGRP-like domain of the PGRP-W polypeptide having the amino acid sequence at positions 52 to 135 in FIGS. 2A-C (SEQ ID NO:4), and/or the predicted PGRP-like domain of the PGRP-C polypeptide having the amino acid sequence at positions 34 to 117 in FIG. 3 (SEQ ID NO:6), or as encoded by the cDNA clones contained in ATCC Numbers 203564, 203563, and 209683, respectively, deposited on Dec. 23, 1998, and Mar. 20, 1998; or as encoded by the cDNA clones contained in ATCC Numbers 203564, 203563, and 209683, respectively, deposited on Dec. 23, 1998, and Mar. 20, 1998; (c) a nucleotide sequence encoding a soluble PGRP-K, PGRP-W, and/or PGRP-C polypeptide having the PGRP-like domain but lacking the leader sequence; and (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), or (d) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), or (d) above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a PGRP-K, a PGRP-W, or a PGRP-C polypeptide having an amino acid sequence in (a), (b), or above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for the production of PGRP-K, PGPR-W, and/or PGRP-C polypeptides or peptides by recombinant techniques.
The invention further provides isolated PGRP-K, PGRP-W, and PGRP-C polypeptides comprising amino acid sequences selected from the group consisting of: (a) the amino acid sequence of the full-length PGRP-K polypeptide having the complete amino acid sequence shown in FIGS. 1A-B (SEQ ID NO:2), the amino acid sequence of the full-length PGRP-W polypeptide having the complete amino acid sequence shown in FIGS. 2A-C (SEQ ID NO:4), the amino acid sequence of the full-length PGRP-C polypeptide having the complete amino acid sequence shown in FIG. 3 (SEQ ID NO:6), or as encoded by the cDNA clones contained in ATCC Numbers 203564, 203563, and 209683, respectively, deposited on Dec. 23, 1998, and Mar. 20, 1998; (b) the amino acid sequence of the predicted PGRP-like domain of the PGRP-K polypeptide having the amino acid sequence at positions 24 to 107 in FIGS. 1A-B (SEQ ID NO:2), the predicted PGRP-like domain of the PGRP-W polypeptide having the amino acid sequence at positions 52 to 135 in FIGS. 2A-C (SEQ ID NO:4), and/or the predicted PGRP-like domain of the PGRP-C polypeptide having the amino acid sequence at positions 34 to 117 in FIG. 3 (SEQ ID NO:6), or as encoded by the cDNA clones contained in ATCC Numbers 203564, 203563, and 209683, respectively, deposited on Dec. 23, 1998, and Mar. 20, 1998; (c) the amino acid sequence of the soluble PGRP-K, PGRP-W, and/or PGRP-C polypeptide having the PGRP-like domain but lacking the leader sequence, wherein each of these domains is defined below.
The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity to those described in (a), (b), or (c) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a PGRP-K, a PGRP-W, or a PGRP-C polypeptide having an amino acid sequence described in (a), (b), or (c) above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a PGRP-K, a PGRP-W, or a PGRP-C polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment, the invention provides an isolated antibody that binds specifically to a polypeptide having an amino acid sequence described in (a), (b), or (c) above.
The invention further provides methods for isolating antibodies that bind specifically to an PGRP-K, PGRP-W, or PGRP-C polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
The invention also provides for pharmaceutical compositions comprising soluble PGRP-K, PGRP-W, and/or PGRP-C polypeptides, particularly human PGRP-K, PGRP-W, and/or PGRP-C polypeptides, which may be employed, for instance, to treat tumor and tumor metastasis, infections by bacteria, viruses and other parasites, immunodeficiencies, inflammatory diseases, regulate the apoptosis and/or proliferation of keratinocytes, epidermal cells, and epithelial cells, mediate antigen processing and presentation, mediate cell activation and proliferation, and are functionally linked as primary mediators of immune recognition and immune responses.
The invention further provides compositions comprising a PGRP-K, PGRP-W, or PGRP-C polynucleotide or a PGRP-K, PGRP-W, or PGRP-C polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise a PGRP-K, PGRP-W, or PGRP-C polynucleotide for expression of a PGRP-K, PGRP-W, or PGRP-C polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of a PGRP-K, PGRP-W, or PGRP-C gene.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by PGRP-K, PGRP-W, or PGRP-C which involves contacting cells which express PGRP-K, PGRP-W, or PGRP-C with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
In another aspect, a method for identifying PGRP-K, PGRP-W, or PGRP-C receptors is provided, as well as a screening assay for agonists and antagonists using such receptors. This assay involves determining the effect a candidate compound has on PGRP-K, PGRP-W, or PGRP-C binding to the PGRP-K, PGRP-W, or PGRP-C receptor. In particular, the method involves contacting a PGRP-K, PGRP-W, or PGRP-C receptor with an PGRP-K, PGRP-W, or PGRP-C polypeptide and a candidate compound and determining whether PGRP-K, PGRP-W, or PGRP-C polypeptide binding to the PGRP-K, PGRP-W, or PGRP-C receptor is increased or decreased due to the presence of the candidate compound. The antagonists may be employed to prevent septic shock, inflammation, and to regulate the growth activity of keratinocytes.
The present inventors have discovered that PGRP-K, PGRP-W, and PGRP-C is expressed in keratinocytes, wound healing tissues, and chondrosarcomas, respectively. For a number of disorders of these tissues and cells, such as tumor and tumor metastasis, infection of bacteria, viruses and other parasites, immunodeficiencies, septic shock, apoptosis or proliferation of these tissues, and proper antigen processing and presentation, it is believed that significantly higher or lower levels of the PGRP-K, PGRP-W, or PGRP-C gene expression can be detected in certain tissues (e.g., keratinocytes, wound-healing tissues, and chondrosarcoma) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a xe2x80x9cstandardxe2x80x9d PGRP-K, PGRP-W, or PGRP-C gene expression level, i.e., the PGRP-K, PGRP-W, or PGRP-C expression level in tissue or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves: (a) assaying PGRP-K, PGRP-W, or PGRP-C gene expression levels in cells or body fluid of an individual; (b) comparing the PGRP-K, PGRP-W, or PGRP-C gene expression level with a standard PGRP-K, PGRP-W, or PGRP-C gene expression level, whereby an increase or decrease in the assayed PGRP-K, PGRP-W, or PGRP-C gene expression level compared to the standard expression level is indicative of a disorder.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of either PGRP-K, PGRP-W, or PGRP-C activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated PGRP-K, PGRP-W, or PGRP-C polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of either PGRP-K, PGRP-W, or PGRP-C activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a PGRP-K, PGRP-W, or PGRP-C antagonist. Preferred antagonists for use in the present invention are either PGRP-K, PGRP-W, or PGRP-C-specific antibodies.